Fermentation processes with low concentrations of carbon-and nitrogen-containing nutrients

ABSTRACT

The present invention describes a fermentation process for the production of a desired compound (such as natamycin) comprising cultivating a filamentous bacterial strain in a liquid fermentation medium, wherein the carbon containing nutrients and nitrogen containing nutrients are maintained at low concentrations in the fermentation medium. The process of the invention reduces the viscosity of the culture medium and therefore increases the yield of the desired compound.

FIELD OF THE INVENTION

The present invention relates to the field of fermentative production ofdesired compounds, such as secondary metabolites, proteins or peptides.

BACKGROUND OF THE INVENTION

The actinomycetes, a family of filamentous bacteria, are of greatimportance for the fermentation industry. Many members of this familyare known to produce secondary metabolites or extracellular enzymes andseveral of these products of bacterial metabolism have an industrialapplication. For obtaining these products, the bacteria are generallycultivated in liquid media (submerged cultures), leading to excretion ofthe products into the liquid, from which they can be isolated. Formationof product can take place during the initial fast growth of the organismand/or during a second period in which the culture is maintained in aslow-growing or non-growing state. The amount of product which is formedper unit of time during such a process (the productivity) is generally afunction of a number of factors: the intrinsic metabolic activity of theorganism; the physiological conditions prevailing in the culture (e.g.pH, temperature and medium composition); and the amount of organismswhich are present in the equipment used for the process. Generally,during optimisation of a fermentation process, it is preferred to obtaina concentration of bacteria that is as high as possible because,assuming that the intrinsic productivity per unit of organism is aconstant, the highest titer of product will be obtained. However, oneparticular characteristic of the bacteria which belong to the family ofactinomycetes, makes it difficult to achieve this goal. Actinomycetes,when grown in submerged culture, have a filamentous morphology, whichgenerally leads to highly viscous culture fluids. A high viscosity ofthe culture limits the rate of oxygen transfer to the culture. Virtuallyall processes utilising actinomycetes depend on the presence andconsumption of oxygen and therefore a limitation in oxygen transfer willimpose a limitation on the overall process productivity. The viscosityof a culture fluid is determined by a number of factors such as thecomposition of the medium, the presence and nature of products excretedby the microorganisms, and (most important) the morphology of themicroorganism. If one could influence the morphological characteristicsof the microorganisms in a positive way (i.e. to decrease the specificviscosity), the process could be operated at a higher production rate ora higher concentration of bacteria could be achieved. Both changes inthe process would result in a higher productivity.

SUMMARY OF THE INVENTION

The present invention provides a fermentation process for the productionof a desired compound comprising culturing a filamentous bacterialstrain in a liquid fermentation medium, wherein the carbon containingnutrients and nitrogen containing nutrients are maintained at lowconcentrations in the fermentation medium.

Preferably, a feed comprising carbon and nitrogen containing nutrientsis supplied to the medium and the nutirients in the feed are in such aratio that low concentrations of both carbon and nitrogen containingnutrients are maintained in the culture.

The filamentous bacteria are preferably of the family Actinomyces, morepreferably of the genus Streptomyces.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has been found that certain medium compositions lead toa reduced culture viscosity of a fermentation process comprising afilamentous bacterial strain, without affecting the production of thedesired compound. An important factor appears to be the ratio ofnitrogen containing nutrients (N) to carbon containing nutrients (C) inthe medium. A high N/C ratio (relative excess of nitrogen compounds)leads to viscous cultures, whereas a low N/C ratio results in relativelylow viscosity of the culture fluid. When the amount of nitrogen in themedium is restricted too much, this leads to very poor growth of theorganism and low amounts of product are formed. However, at anintermediate N/C ratio, growth of the organism is good and productformation is normal, while the morphology of the organism is apparentlychanged in such a way that the viscosity of the culture fluid issignificantly reduced. The consequence of this finding is, that bycarefully controlling the medium, or more specifically by controllingthe ratio of carbon and nitrogen containing nutrients in the medium, aprocess comprising a filamentous bacterial strain can be improvedsignificantly.

Bacterial strains of the family Actinomycetes are known to producedesired compounds, which have commercial applications, such as secondarymetabolites, proteins and peptides. Examples thereof are natamycin,nistatine, glucose isomerase and clavulanic acid.

For example, the actinomycetes strains Streptomyces natalensis andStreptomyces silvosporens produce the antifungal compound natamycin,which has several applications as an antifungal compound. Fermentationprocesses comprising such filamentous bacteria are generallycharacterised by two phases. Usually the process starts with a phasewhere growth of the microorganism occurs until conditions for growthbecome unfavourable, for instance because one of the growth supportingnutrients becomes depleted from the medium. The initial (batch) phasemay be followed by a phase where the microorganisms are maintained in aviable state. Often most of the product of interest is formed in thissecond phase. In this second phase, more nutrients may be supplied tothe culture, either discontinuously as a single or repeated charge offresh nutrients, or continuously by feeding one or more nutrientscontaining fluids in to the fermentation vessel. This mode offermentation is called fed-batch fermentation. Preferably, afermentation process may be further prolonged by removing part of thefermentation mash, for instance when the fermentation vessel becomescompletely filled as a result of feeding with nutrient containingfluids. This process form is called extended fermentation or repeated(fed-)batch fermentation.

The initial (batch) phase will end when one of the nutrients isdepleted. This phase may be followed by measuring the oxygen uptakewhich will decrease towards the end of the initial phase. In general,the initial phase will take 6 to 48 hours. The second phase starts whenfeeding of the nutrients is started. Feeding of nutrients allows thecontinuation of the fermentation process for a longer period than ispossible in simple batch fermentation process.

In general, for each production process, the optimal ratio of carbon andnitrogen containing nutrients can be determined by the skilled person,depending on the elementary composition of the organism and theproduct(s), the effect of the N/C ratio on the physiology of theorganism and, more specifically, the product forming capacity of theorganism. It has been found that neither carbon excess nor nitrogenexcess will lead to the desired result. In the optimal situation, boththe available carbon and nitrogen will be almost depleted from themedium at the end of the batch process and/or during the process ofprolonged fed-batch type fermentation. The concentration of the nitrogencontaining nutrient in the medium during the second phase is preferablyless than 0.5 g/l, more preferably less than 0.25 g/l and mostpreferably less than 0.1 g/l (expressed as gram of nitrogen per litre).The concentration of the carbon containing nutrient is preferably lessthan 5 g/l, more preferably less than 2.5 g/l and most preferably lessthan 1 g/l (expressed as gram of carbon per litre). The feed can besupplied as one feed containing all the nutrients or preferably as morethan one subfeeds each comprising either a nitrogen containing nutrient,a carbon containing nutrient or a combination of nitrogen and carboncontaining nutrients.

The feed is also controlled in such a way that the amount of oxygen isbetween 20 and 70% of air saturation, preferably between 30 and 60% ofair saturation.

Oxygen, typically in the form of air, is generally introduced at or nearthe bottom of the fermentor. One of more nozzles are installed for theintroduction of air or another oxygen containing gas such as (purified)oxygen.

Optionally, a stirrer is present in the reactor to stimulate the oxygenuptake. Moreover, the stirrer prevents concentration gradients of thefeed or subfeed developing in the fermentor.

LEGEND TO THE FIGURES

FIG. 1: Viscosity development of a nitrogen excess-culture (●) and anitrogen-carbon double-limited culture (♦).

FIG. 2: Agitation power required to control the dissolved oxygenconcentration at a 30% air saturation. Both cultures, nitrogen excess(∘) and nitrogen-carbon double-limited (⋄), were operated underotherwise similar process conditions.

FIG. 3: Viscosity development of a nitrogen excess culture (∘) and anitrogen-carbon double-limited culture (♦).

FIG. 4: Product accumulation in a nitrogen excess culture (∘) and anitrogen-carbon double-limited culture (♦).

FIG. 5: Full scale fermentation of Streptomyces natalensis to producenatamycin. The initial process (●) used a limiting feed of soybean oil,while the NH3 concentration was kept at a non-limiting level. In theimproved process (♦) the NH3 concentration was kept at a low value bycontinuous feeding of a NH3 solution in proportion to the oil feedingrate. The reduced culture viscosity allowed faster feeding of oil. Theincrease in product formation was approximately proportional to theincrease in oil feeding rate.

EXAMPLES Example 1

Steptomyces natalensis strain ATCC27448 was cultivated in 2000 mlconical shake containing 500 mL growth medium of the followingcomposition: g/L Glucose.1H₂O 30 Casein hydrolysate 15 Yeast Extract(dried) 10 De-foamer Basildon 0.4

The pH was adjusted to 7.0 by adding NaOH/H₂SO₄, and the medium wassterilized by autoclavation (20 minutes at 120° C.). The content of afull-grown shake flask was used to inoculate a fermentation vesselcontaining 6 L medium of the following composition: 9/L Soybean flower25 Soybean oil 8 Corn Steep (dried) 1 KH₂PO₄ 0.45 Trace element solution17 De-foamer Basildon 0.4

The composition of the trace element solution was as follows: g/L Citricacid.1H₂O 175 FeSO₄.7H₂O 5.5 MgSO₄.7H₂O 100 H₃BO₃ 0.06 CuSO₄.5H₂O 0.13ZnSO₄.7H₂O 1.3 CoSO₄.7H₂O 0.14

The temperature and pH of the medium were controlled at 25° C. and 7.0respectively. Dissolved oxygen concentration was kept above 30% of airsaturation, by increasing airflow and/or stirrer speed when necessary.After preliminary growth in batch culture for approximately 24 hours theculture entered the second phase of fermentation. During the secondphase, the growth and product formation were continued by feeding puresoybean oil. A second feeding line was installed to feed ammonia. Theaverage feeding rate of the soybean oil was 3 g/h. Ammonia was suppliedin proportion to the soybean oil feeding rate. A series of fermentationswere carried out, in which different ammonia feeding rates were appliedwhile keeping the soybean oil feeding rate constant. For this strain,the carbon source and the nitrogen source were totally consumed when theratio of NH3 to oil was in the range of 30-40 mg NH3/g oil. Thiscondition of C—N double limitation resulted in cultures with the lowestspecific viscosities. Nitrogen excess (NH3/oil ratio>40 mg/g) resultedin a considerable increased viscosity of the culture. Carbon excess(NH3/oil ratio<30 mg/g) had a similar effect. In addition, theaccumulation of oil had a negative effect on the culture viability. Therange of ratios of nitrogen containing nutrients versus carboncontaining nutrients is dependent on the strain and the nature of thenitrogen and carbon sources. For every new process, the optimal rangecan therefore be determined by the present procedure.

Two experiments were carried out according to the process describedabove. One experiment was aimed to reach a condition of nitrogen excess(i.e. the culture is then purely limited by the soybean oil feedingrate). In another experiment the rate of ammonia feeding relative tosoybean oil feeding was reduced, in order to arrive at a condition wherethe concentration of both nutrients (soybean oil and ammonia) in thefermenter vessel is very low. For the test organism (Strepromycesnatalensis) in the chosen conditions, the ratio of ammonia feeding raterelative to the oil-feeding rate should be around 35 mg NH₃ per g oil.The ammonia surplus experiment was carried out at a ratio of 45 mg NH₃per g oil.

The effect of the carbon-nitrogen double limitation is clearlydemonstrated in FIG. 1. Under nitrogen excess conditions the viscosityreaches the usual high values. Under conditions of simultaneous carbonand nitrogen limitation, the viscosity drops to a much lower value,causing better aeration conditions. For a good production it ispreferred that the dissolved oxygen concentration is maintained at alevel of above 30% of air saturation. FIG. 2 illustrates that formaintaining this dissolved oxygen concentration much less agitationpower (energy) is needed when the culture is under a condition ofnitrogen-carbon double limitation.

Example 2

Another fermentation experiment was carried out using the same procedureas described in Example 1 using a strain of Streptomyces natalensis.This strain is a producer of the anti-fungal compound natamycin. In thisexperiment two fermentations were run. One experiment was under carbonlimitation and nitrogen excess (NH₃ level was kept at 150-200 mg/Lduring the oil feeding phase). The second experiment was run undernitrogen-carbon double limitation during the oil feeding phase,employing a NH/oil ratio of 32 mg/g. Some results are shown in FIGS. 3and 4. It is obvious that a very significant difference in viscosity wasobserved between the two modes of fermentation. A low viscosity is verybeneficial for efficient process operation. However, a low viscositycoupled with a poor product formation potency would be negative. In thisexperiment, the product formation was not affected at all by theconditions leading to low viscosity (FIG. 3). The rate of productformation in the nitrogen-carbon double limitation experiment is fasterin the second part of the fermentation despite a slightly slower start.

Example 3

The information obtained in the experiments described in Examples 1 and2 was used to improve the actual production process of natamycin on anindustrial scale (100 m³ scale). The reduced viscosity allowsintensification of the process by faster feeding of the main nutrientsoybean oil. The feeding rate of NH3 was proportional to the feeding ofoil, as described in the Examples 1 and 2, resulting in carbon-nitrogendouble limitation during the feeding phase (which started at about 24hours after inoculation of the fermentation vessel). The processconditions and medium composition were similar to the small scaleexperiments described in Examples 1 and 2. Starting with a smallincrease, the oil feeding rate was increased step-wise from run to run,until a process intensity was reached which could just be maintained onminimal dissolved oxygen tension. FIG. 5 illustrates, the improvement inproduct output resulting from the higher oil feeding rate was quitesubstantial.

1. A fermentation process for the production of a desired compoundcomprising cultivating a filamentous bacterial strain in a liquidfermentation medium, wherein the carbon containing nutrients andnitrogen containing nutrients are maintained at low concentrations inthe fermentation medium.
 2. A fermentation process according to claim 1,wherein the concentration of the nitrogen containing nutrient in themedium is less than 0.5 g/l (expressed as gram of nitrogen per litre).3. A fermentation process according to claim 1, wherein theconcentration of the carbon containing nutrient in the medium is lessthan 5 g/l (expressed as gram of carbon per litre).
 4. A fermentationprocess according to claim 1, wherein a feed comprising carboncontaining nutrients and nitrogen containing nutrients is supplied tothe medium and wherein the nutrients in the feed are in such a ratiothat low concentrations of both carbon and nitrogen containing nutrientsare maintained in the culture.
 5. A fermentation process of claim 1,wherein the feed is supplied to the medium via more than one subfeed andwherein each subfeed comprises nitrogen containing nutrients, carboncontaining nutrients or a combination of nitrogen and carbon containingnutrients.
 6. A fermentation process according to claim 1, wherein theamount of oxygen in the medium is between 20 and 70% of air saturation.7. A fermentation process according to claim 6, wherein the amount ofoxygen in the medium is between 30 and 60% of air saturation.
 8. Aprocess according claim 1, wherein the bacteria are of the family ofActinomycetes.
 9. A process according to any claim 8, wherein thebacteria are of the genus Streptomyces.
 10. A process according to claim9, wherein the bacteria are Streptomyces natalensis or Streptomycesgilvosporeus and wherein the desired compound is natamycin.
 11. Afermentation process according to claim 1, wherein the carbon containingnutrient is for more than 50% soybean oil (calculated as gram of carbon)and the nitrogen containing nutrient is for more than 50% ammonia(calculated as gram of nitrogen).